The alphavirus, Sindbis virus, provides a valuable model for examining interactions between RNA and protein in required for encapsidation of a viral genome and for studying the process of replication of viral RNA - the subjects of this research program. We had analyzed a series of deletions in Sindbis defective interfering (DI) RNAs derived from cDNAs and demonstrated that only sequences in the 5' and 3' terminal regions are essential for these genomes to be replicated and encapsidated. The ability to transcribe Sindbis DI RNAs, infectious virion RNA and almost any modified form of these molecules from cloned cDNAs opens new avenues for investigating the replication and assembly of these RNAs. Our studies on encapsidation will be directed toward identifying sequences in the viral RNAs required for specific interactions with the capsid protein and determining which domain(s) of the capsid protein are involved in this interaction. We also will attempt to identify an assembly initiation complex. The overall goals of our studies on replication will be to define the essential sequences in the RNA that must be recognized by the nonstructural (replicase) proteins and to dissect the steps in the replication process. We plan to devote a major effort to preparing large amounts of the nonstructural proteins using expression vectors. This should facilitate our efforts to develop in vitro assays.